3'-phosphoinositide-dependent kinase-1 (PDK-1) is a 65 kDa Ser/Thr protein kinase that is activated downstream of PI3-kinase in the insulin signaling pathway. While PDK-1 has been shown to play an important role in insulin signaling, how the kinase is regulated in cells remains largely unknown. Using pharmacological inhibitors, I have found that insulin-stimulated phosphorylation of PDK-1 was inhibited by PI3-kinase and PKC inhibitors in cells. In addition, PKCzeta, a direct substrate of PDK-1 that has been shown to be involved in insulin-stimulated glucose uptake via GLUT4, can phosphorylate kinase-dead PDK-1 in vitro on a novel serine residue(s). This is the first time a substrate of PDK-1 is demonstrated to phosphorylate this central kinase. The PKCzeta-mediated phosphorylation of PDK-1 could represent a negative feedback mechanism which results in down-regulation of PDK-1 activity. To further characterize the phosphorylation of PDK-1, I will: 1) identify the novel site(s) of phosphorylation on PDK-1 by PKCzeta; and 2) determine whether PKC zeta-mediated phosphorylation affects PDK-1 activity and function in cells. [unreadable] [unreadable]